Berry preparations and extracts

ABSTRACT

Compositions having antioxidant and anti-inflammatory activity, and methods for providing such compositions, are disclosed. In one aspect, the compositions are derived by exposing a berry to an acidic solvent composition, adding a cryoprotectant, and recovering a berry extract having a stabilized anthocyanin content. Compositions comprising the stabilized anthocyanin-containing berry extract, formulated for oral and/or topical administration, are provided also.

This application is a continuation-in-part utility patent applicationwhich claims priority to U.S. patent application Ser. No. 12/088,156filed on Mar. 26, 2008, which in turn is a national stage application ofinternational patent appl. no. PCT/US06/37237 filed on Sep. 26, 2006 andclaiming the benefit of priority in U.S. Provisional Patent Appl. Ser.No. 60/720,892 filed on Sep. 27, 2005, the disclosures of each of whichare incorporated in their entirety herein by reference.

TECHNICAL FIELD

The present invention relates to compositions having antioxidant andanti-inflammatory activity, derived from a berry. In particular, theinvention relates to a method for deriving an extract having antioxidantand anti-inflammatory activity from a berry, and to formulatedcompositions derived by the method for oral and topical administration.Still further, the invention provides methods and compositions fortreatment of inflammation, oxidative stress, or cancer comprisingadministering an effective amount of the composition of the invention.

BACKGROUND OF THE INVENTION

Polyphenolics (also known as phenolics) are metabolites found in plants,fruits, and vegetables. Phenolics have a number of functions includingacting as free radical scavengers. Thus, phenolics disrupt manybiological processes. The most notable type of phenolics are flavonoidswhich consist of proanthocyanidins, anthocyanidins, flavones, flavonolsand their glycosides (Macheix, 1990). Anthocyanins are responsible forthe red, purple, and blue colors of many fruits and vegetables. The termanthocyanin refers to a group of pigments found in plants, fruits, andvegetables that can be classified as both flavonoid and phenolic.Anthocyanins are not found in animals, microorganisms, or marine plants.It is thought that anthocyanins function by attracting insects topromote pollination but also to protect the plants from damage caused byultraviolet (UV) radiation. Anthocyanins are glycosides of polyhydroxyland polymethoxyl derivatives of 2-phenylbenzopyrylium or flavyliumsalts, and are soluble in water. The anthocyanins are electrondeficient, and are therefore are strong scavengers for reactive oxygenspecies (ROS) such as free radicals. Although there are hundreds ofdifferent anthocyanins found in nature, six anthocyanin compoundspredominate including: delphinidin, petunidin, cyanidin, pelargonidin,peonidin, and malvidin. The daily intake of anthocyanins in the UnitedStates is about 180-215 mg/day, and constitute the largest intake ofphenolic compounds (Hertog, 1993).

Anthocyanins originally drew interest due to their role in colordegradation in fruits, and their potential use as natural foodcolorants. More recently, anthocyanins have received attention becauseof their possible health benefits as natural antioxidant andanti-inflammatory compounds, and potentially as anti-cancer compounds.Antioxidant property, effects, or activity refers to compositions thatinhibit, reduce, or reverse oxidation or the effects of oxidation, suchas for example the oxidative process caused by free radicals. A freeradical may be any chemical species that includes one or more unpairedelectrons, and without limitations includes chemicals such as hydroxylradical, superoxide radical, nitric oxide, and nitrogen dioxide.Examples of conditions involving free radical oxidative damage include,but are not limited to, aging, disease, stress, ultraviolet radiation,exercise, cancer, smoking, atherosclerosis, and chronic inflammation.Studies showed that antioxidant activity of cyanidins was greater thanthat of vitamin E and Trolox, and comparable to that of butylatedhydroxytoluene (BHT) and butylated hydroxyanisole (BHA) (Wang et al.,1997; Rice-Evans et al., 1995; Liu et al., 2002; Proteggente et al.,2002; Wang et al., 1999).

Inflammation, or the biological state of being inflamed, is generallycharacterized by pain, redness, and swelling, and may result fromphysical causes such as injury, chemical causes such as exogenoussubstances including toxins, or biological causes such as infection by avirus, a bacteria, a parasite, or other disease-causing agent. Asexamples, various conditions associated with inflammation of thegastrointestinal tract are known, including but not limited to acute orchronic conditions or diseases such as inflammatory bowel disease,gastroesophageal reflux disease, diarrhea, radiation-induced enteritis,chemotherapy-induced enteritis, Crohn's disease, irritable bowelsyndrome, diverticulitis, ulcers, colitis, viral infection, bacterialinfection, and parasitic infection or infestation. In individualsafflicted with the above and similar conditions, affected cells such asdendritic cells, monocytes, macrophages, fibroblasts, endothelial cells,and T cells release causative agents, including cytokines such asinterleukins (IL), tumor necrosis factor (TNF), interferons (IFN), andthe like which trigger inflammation and/or an inflammatory response.Cytokines important in inflammation include, but are not limited to,IL-1, IL-6, IL-12, TNF-α, and IFN-α. Anti-inflammatory properties,effects, or activity refers to reducing inflammation and promotinghealing of cells and tissues subject to inflammation and/or inflammatoryprocesses.

Anti-cancer property, effect, or activity refers to a property of asubstance, chemical, or material that can slow the proliferation oftumor cells (termed anti-proliferative effect), or kill tumor cells(termed cytotoxic effect). Recent studies have shown also that berriessuch as black raspberries possess cancer-preventing properties at bothin-vitro and in-vivo levels (Kresty et al., 2001; Castro et al., 2002;Xue et al., 2001; Huang et al., 2002; Rodrigo et al, in press; U.S.patent application Ser. No. 10/951,413). In particular, anthocyaninshave been shown to demonstrate a wealth of chemopreventive properties(Hecht et al., in press; Liu et al., 2002; Katsube et al., 2003; Hu etal., 2003).

However, anthocyanin compounds are inherently unstable both in vivo andin vitro, and tend to degrade over time, negating any health or medicalbenefits associated therewith (Rubinskiene et al., 2005; Nielsen et al.,2003; Morais et al., 2002). A need in the art therefore exists formethods for preparing compositions from anthocyanin-containing fruitssuch as berries which provide a stable anthocyanin content. Stillfurther, there is a need for compositions including such stableanthocyanin compositions to provide health and medical benefits toindividuals utilizing them. In particular, compositions formulated fororal administration and topical administration are described herein.However, it will be appreciated that other formulations are contemplatedand can be derived by the skilled artisan from the teachings hereinusing methods known in the art, including injectable formulations.Accordingly, the present invention contemplates also injectableformulations including, but not limited to, solutions, suspensions,emulsion, microemulsions, micelles, liposomes, nanoparticles,microparticles, implants, depots, and polymer conjugates.

SUMMARY OF THE INVENTION

The present invention provides methods for deriving stable compositionsfrom blackberries, and compositions incorporating such stablecompositions. In one aspect, a method is provided for preparing acomposition having antioxidant and anti-inflammatory activity,comprising exposing a berry to a solvent composition having a pH of fromabout 1 to about 3, adding a cryoprotectant, and recovering a berryextract having a stable anthocyanin content. The berry may be ablackberry. In one embodiment, the method comprises the steps ofphysically disrupting a quantity of berries, exposing the physicallydisrupted berries to the solvent composition, recovering a berry extracthaving a pH of from about 1.0 to about 4.5, and adding a cryoprotectant.The cryoprotectant is added in an amount of at least 2:1 (w/w)cryoprotectant:berry extract. The berry extract so provided typicallycomprises at least one stable anthocyanin in an amount effective toprovide antioxidant and anti-inflammatory activity. In anotherembodiment, the recovered berry extract has a pH of about 3.5. Thephysically disrupted berries may be dewatered prior to being exposed tothe solvent composition, after being exposed to the solvent composition,or both.

In another aspect, the present invention provides stabilizedcompositions having antioxidant and anti-inflammatory activity, preparedby the method of the invention as described above. In one embodiment, acomposition is provided having antioxidant and anti-inflammatoryactivity, comprising a berry extract having at least one stableanthocyanin in an amount effective to provide antioxidant andanti-inflammatory activity. The berry extract is provided by the stepsof physically disrupting a quantity of berries and exposing thephysically disrupted berries to a solvent composition having a pH offrom about 1 to about 3, and adding a cryoprotectant, which stabilizesthe at least one anthocyanin. The cryoprotectant is added in an amountof at least 2:1 (w/w) cryoprotectant:berry extract. The berry extractmay be derived from a blackberry. Compositions comprising the berryextract of the present invention, formulated for oral and/or topicaladministration, are provided also, including without limitationnutritional supplements, capsules, tablets, chewing gum, lotions,creams, mucoadhesive gels, vanishing lotions, vanishing creams, and thelike.

In yet another aspect, the present invention provides methods fortreatment of inflammation, oxidative stress, or cancer in a mammal inneed thereof, comprising administering an effective amount of thecomposition of the invention. Still yet further, another aspect of theinvention is compositions comprising a therapeutically effective amountof the composition of the invention, formulated in a pharmaceuticallyacceptable vehicle. The composition may be provided in combination withadditional anticancer agents, additional anti-oxidant agents, and/oradditional anti-inflammatory agents.

As should be appreciated, the embodiments shown and described herein arean illustration of one of the modes best suited to carry out theinvention. It will be realized that the invention is capable of otherdifferent embodiments and its several details are capable ofmodification in various, obvious aspects all without departing from theinvention. Accordingly, the drawings and descriptions will be regardedas illustrative in nature, and not as restrictive. Unless otherwiseindicated, all patents, patent applications, and non-patent documentsreferenced in the present disclosure are incorporated herein byreference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings incorporated in and forming a part of thespecification, illustrate several aspects of the present invention andtogether with the description serve to explain the principles of theinvention. In the drawings:

FIG. 1 shows percent weight increase of active blackberry powder as afunction of time when stored at 25° C. and at 4° C.;

FIG. 2 depicts a standard HPLC chromatogram of a blackberry extract madeaccording to the present invention;

FIGS. 3A and 3B show the stability of anthocyanins as a function of pHand time;

FIG. 4 shows the stability of a specific anthocyanin(cyanidin-3-glucoside) in a blackberry preparation made according to thepresent invention as a function of time and storage condition;

FIGS. 5A and 5B show the stability of anthocyanins in blackberrypreparations made according to the present invention as a function oftime and temperature;

FIG. 6 presents a dissolution profile of enteric-coated hard capsulescontaining a berry composition according to the present invention;

FIG. 7 shows a dissolution profile of enteric-coated hard capsulescontaining a berry extract according to the present invention;

FIG. 8 shows inhibition of proliferation of HT-29 cells by a blackberryextract according to the present invention; and

FIG. 9 shows inhibition of IL-12 release from murine dendritic cells bya blackberry extract according to the present invention.

Reference will now be made in detail to the present preferred embodimentof the invention, an example of which is illustrated in the accompanyingdrawings.

DETAILED DESCRIPTION OF THE INVENTION

Blackberries and raspberries, sometimes referred to as “brambles”, are aspecies of fruit in the genus Rubus. Rubus is quite diverse and has 12subgenera with several of the subgenera having more than a hundredspecies. In the United States, R. allegheniensis, R. argutus, R.cuneifolius, R. Canadensis, and R. trivialis are the dominant Rubusspecies. Blackberries belong to the subgenus Eubatus, and have muchgreater complexity than raspberries in terms of genetic background,growth characteristics, and number of species. Blackberries are nativeto Asia, Europe, North and South America. They have been grown in Europefor over 2000 years as food and for medical applications. Blackberriesgrow in three different forms: trailing, semi-erect, and erect. In theeastern half of the United States, the dominate form is the erectincluding the thorny types, Cherokee, Comanche, Cheyenne, Darrow, IlliniHardy Blackberry, Shawnee, and Choctaw. The thornless cultivars includeHull, Chester, Navaho, and Arapaho. Other types of blackberries include,but are not limited to, Eldorado, Ebony King, Raven, Ranger, Hedrick,Bailey, and Brazos. Semi-erect blackberries requiring a trellis forproduction include Black Satin, Chester, Dirksen, Hull Thornless,Smoothstem, and Thornfree. In the Pacific Northwest, blackberries areoften called Marionberries. Other berries that have genetic similarityto blackberries include Boysenberry, Loganberry, Olallieberry, andYoungberry. A 2002 report by the USDA lists the total US production ofall blackberry types to be about 56 million pounds grown on 8370 acres.According to the USDA, of the blackberries harvested in the US, about40% are used for jams and jellies, 25% for bakery products, 23% frozenor canned, and the 12% for juices or other food.

Tsao et al. (2003) showed that, relative to other fruits, blackberrieshave high concentrations of polyphenolics such as anthocyanins with asmuch as 1923 μg/g wet fruit. Sellappan et al. (2002) showed thatblackberries had high concentrations of ellagic acid; Chocktaw had 33.81mg/100 g berry and Kiowa had 30.01 mg/100 g berry. Blackberries werealso found to contain flavonoids, with the major flavonoid, catechin,present in Choctaw at 312.86 mg/100 g berry. Sellappan showed that theaverage total anthocyanin and polyphenolic content in blackberries was116.59±8.58 mg/100 g berry and 486.53±97.13 mg/100 g berry,respectively.

Accordingly, in one embodiment the present invention describes methodsfor deriving stable compositions from blackberries, and compositionsmade by those methods. However, it will easily be understood by thoseskilled in the art that the methods and compositions taught in thisinvention apply to many other types of fruits, plants, and vegetableshaving an anthocyanin content, including but not limited to:strawberries, black raspberries, apple, crabapple, chokeberry, Hawthorn,Juneberry, Loquat, pear, apricot, cherry, plum, peach, various otherraspberries, cloudbeny, wineberry, salmonberry, bearberry, bilberry,cranberry, huckleberry, barberry, currant, elderberry, gooseberry,hackberry, honeysuckle, nannyberry, sheepberry, sea grape, wolfberry,crowbeny, goumi, kiwi, various other grapes, thimbleberry, salmonberry,various types of melons such as watermelon, fig, lime, avocado, feijoa,guava, kumquat, longan, lychee, passion fruit, and pineapple.

In one aspect, the present invention provides a method for preparing acomposition having antioxidant and anti-inflammatory activity,comprising exposing a berry to a solvent composition having a pH of fromabout 1 to about 3, adding a cryoprotectant, and recovering a berryextract having a stabilized anthocyanin content. As discussed above, theberry may be a blackberry. In one embodiment, the method comprises thesteps of: physically disrupting a quantity of berries, exposing thephysically disrupted berries to the solvent composition, recovering aberry extract having a pH of from about 1.0 to about 4.5, and adding acryoprotectant. Suitable cryoprotectants include, but are not limitedto, monosaccharides such as glucose, fructose, maltose, ribose, mannose,and xylose, disaccharides such as trehalose, sucrose, myoinositol,phosphorylated inositols, and glycerol, polysaccharides such ashydroxyethyl starch and other starches, dextran, and hyaluronic acid,and polymers such as polyvinylpyrrolidone, alginates, carrageenan,cyclodextrins, polyvinyl alcohol, cellulose-derivatives such ascarboxymethyl cellulose, methyl cellulose, hydroxypropyl cellulose,hydroxypropylmethyl cellulose, xanthan gum, and chitosan. It will beappreciated that the final pH of the extract will depend on both theconcentration of the berry extract in the solvent composition and on thespecific solvent composition used for extraction. The berry extract sorecovered typically comprises at least one stabilized anthocyanin in anamount effective to provide antioxidant and anti-inflammatory activity.In another embodiment, the recovered berry extract has a pH of about3.5. The stabilized anthocyanin may be a delphinidin, a petunidin, acyanidin, a pelargonidin, a peonidin, a malvidin, or any combinationthereof.

The physically disrupted berries may be exposed to a solvent compositioncomprising an alcohol and at least one acid, with the acid provided in asufficient amount whereby the solvent composition has a pH of from about1 to about 3. The acid may be provided in an amount of from about 0.005%to about 3% (v/v) of the alcohol. In one embodiment, the acid isprovided in an amount of from about 0.01% to about 2% of the alcohol(v/v). In yet another embodiment, the acid is provided from about 0.01%to about 1% of the alcohol (v/v). Any of a number of alcohols may besuitable for the present invention, including, but not limited to, lowerchain alcohols such as methanol, ethanol, propanol, butanol, andmixtures thereof. The acid may be any acid suitable for providing thedesired pH for the solvent composition, including, but not limited, tohydrochloric acid, acetic acid, citric acid, lactic acid,trifluoroacetic acid, aspartic acid, glutamic acid, sulfur-containingacids such as sulfonic acid, phosphoric acid, maleic acid, and mixturesthereof. In yet another embodiment, the alcohol can be replaced with asuitable ketone or a suitable halogenated olefin, such as acetone,chloroform, methylene chloride, or mixtures thereof.

The physically disrupted berries may be dewatered to a residual watercontent of up to 20% (w/v) prior to being exposed to the solventcomposition, after being exposed to the solvent composition, or both. Inone embodiment, the physically disrupted berries are dewatered to aresidual water content of up to 10% (w/v). It will be appreciated thatany suitable method may be employed for dewatering, such as heating orcentrifuging a berry puree to remove water, followed by crushing thedried berry puree to a powder. Typically, a powder in accordance withthe present invention is prepared by lyophilization or freeze-drying,i.e., freezing followed by removal of water by sublimation.Beneficially, freeze-drying provides a chemically and physically stable,free-flowing dry powder.

In another aspect, the present invention provides compositions havingantioxidant and anti-inflammatory activity, prepared by the method ofthe invention as described above. In one embodiment, a composition isprovided having antioxidant and anti-inflammatory activity, comprising aberry extract having at least one stabilized anthocyanin in an amounteffective to provide antioxidant and anti-inflammatory activity. Theberry extract may be provided by the steps of physically disrupting aquantity of berries, exposing the physically disrupted berries to asolvent composition, and adding a cryoprotectant, which stabilizes theat least one anthocyanin. Suitable cryoprotectants include, but are notlimited to, monosaccharides such as glucose, fructose, maltose, ribose,mannose, and xylose, disaccharides such as trehalose, sucrose,myoinositol, phosphorylated inositols, and glycerol, polysaccharidessuch as hydroxyethyl starch and other starches, dextran, and hyaluronicacid, and polymers such as polyvinylpyrrolidone, alginates, carrageenan,cyclodextrins, polyvinyl alcohol, cellulose-derivatives such ascarboxymethyl cellulose, methyl cellulose, hydroxypropyl cellulose,hydroxypropylmethyl cellulose, xanthan gum, and chitosan. The berryextract may be derived from a blackberry. In one embodiment, the berryextract has a pH of from about 1.0 to about 4.5. In another embodiment,the berry extract has a pH of about 3.5. The stabilized anthocyanin maybe a delphinidin, a petunidin, a cyanidin, a pelargonidin, a peonidin, amalvidin, and combinations thereof.

The solvent composition may comprise an alcohol and at least one acid,wherein the acid is provided in an amount sufficient wherein a pH of thesolvent composition is from about 1 to about 3. The acid may be providedin an amount of from about 0.005% to about 3% (v/v) of the alcohol. Thealcohol may be any suitable alcohol, including but not limited to lowerchain alcohols such as methanol, ethanol, propanol, butanol, andmixtures thereof. The acid may be any acid suitable for providing thedesired pH of the solvent composition, including, but not limited to,hydrochloric acid, acetic acid, citric acid, lactic acid,trifluoroacetic acid, aspartic acid, glutamic acid, formic acid,phosphoric acid, maleic acid, and combinations thereof. Optionally, inplace of the alcohol a suitable ketone or a suitable halogenated olefinmay be used, such as acetone, chloroform, methylene chloride, andmixtures thereof. As discussed above, the physically disrupted berriesmay be dewatered to a water content of up to 20% (w/v), such as byfreeze-drying, either prior to exposing to the solvent composition,after exposing to the solvent composition, or both.

A variety of formulations for oral and/or topical administration arecontemplated for the composition of the present invention. In oneembodiment, a formulation for oral administration is provided,comprising the berry extract in an amount of from about 3% (w/w) toabout 90% (w/w). In another embodiment, a formulation for topicaladministration is provided, comprising the berry extract in an amount offrom about 1% (w/w) to about 20% (w/w). It will be appreciated by theskilled artisan that a large number of topical and oral formulations areknown in the art, such as nutritional supplements, capsules,enteric-coated capsules, film-coated capsules, tablets, enteric-coatedtablets, film-coated tablets, chewing gums, lotions, creams,mucoadhesive gels, vanishing lotions, vanishing creams, and the like.Other ways to apply a composition topically formulations are known,including but not limited to: sprays, ointments, gels, patches, andneedle-free devices that deliver their contents by diffusion, mechanicalor gas-driven energy. The making of such formulations and/or devices iswell within the ability of the skilled artisan, and such formulationsand methods are contemplated also by the present invention.

For example, it is known to provide compositions in emulsion ormicroemulsion form, as lotions and/or creams. A lotion refers to asemi-viscous emulsion that is meant to be applied to the skin. A creamrefers to a more viscous emulsion that is also meant to be applied tothe skin. Lotions and creams may be of the oil-in-water or water-in-oiltype. In one embodiment of the present invention, lotions and creams ofthe oil-in-water type are provided which stabilize a compositionaccording to the present invention, and/or promote its penetration intoor through the skin layers.

An emulsion refers to a biphasic opaque mixture of two immiscibleliquids stabilized by a surfactant. Emulsions are thermodynamicallyunstable systems, and usually require the application of high-torquemechanical mixing or homogenization to produce dispersed droplets in therange of about 0.2 to 25 μm. In contrast, a microemulsion is a stablebiphasic mixture of two immiscible liquids stabilized by a surfactantand usually a co-surfactant. Microemulsions are thermodynamicallystable, isotropically clear, form spontaneously without excessivemixing, and have dispersed droplets in the range of about 5 nm to 140nm. Both microemulsions and emulsions can be made as water-in-oil oroil-in-water systems. In a water-in-oil system, the dispersed phase iswater and the continuous phase is oil. In an oil-in-water system, thedispersed phase is oil and the continuous phase is water. Whetherwater-in-oil or oil-in-water systems will form is largely influenced bythe properties of the surfactant. The use of surfactants that havehydrophilic-lipophilic balances (HLB) of about 3-6 tend to promote theformation of water-in-oil microemulsions while those with HLB values ofabout 8-18 tend to promote the formation of oil-in-water microemulsions.

Formulations contemplated for oral administration directly to thegastrointestinal tract include, but are not limited to, nutritionalsupplements, capsules, enteric-coated capsules, film-coated capsules,tablets, enteric-coated tablets, film-coated tablets, chewing gums,caplets, solutions, and suspensions. It is envisioned that more novelparticle-based suspensions for oral administration can be produced, suchas emulsions, microemulsions, nanoparticle or microparticle suspensions.It is also contemplated in this invention that a formulation for oraladministration may be prepared that is delivered to and retained in themouth, whereby the contents and/or the active ingredient primarily draininto the stomach. Formulations meeting these criteria include, but arenot limited to, chewing gums, gels, sprays, lozenges, lollipops or othercandies, rapidly dissolving strips, and the like. In one embodiment, theformulation for oral administration is a tablet or hard-gelatin capsule.In another embodiment, the formulation is coated with a substance tocontrol the rate or location of disintegration, i.e., an enteric-coatedor film-coated formulation.

The present invention contemplates providing enteric-coated oralformulations. An enteric-coating refers to a coating on an oral dosageform that is meant to cause the oral dosage form to remain intact in thelower pH of the stomach, but disintegrate once the dosage form reachesthe higher pH of the intestines including the small intestines, largeintestines, colon, or rectum. The most common enteric coatings are thosethat remain intact (undissociated) in the low pH of the stomach, butionize when the pH is above pH 4-5, pH 5-7, or even above pH 7. Commonenteric-coating materials include, but are not limited to, celluloseacetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate, cellulose acetate trimellitate, carboxymethylethylcellulose, and hydroxypropyl methylcellulose acetate succinate, andpolymethacrylates (methacrylic acid copolymer) including EUDRAGIT-basedpolymers. It is also contemplated to provide film-coated oralformulations. A film-coating typically targets the coated substance tothe colon by remaining undissociated or stable until a pH 7 environmentis reached. Film-coating materials to achieve this goal include, but arenot limited to, EUDRAGIT-based polymers that are soluble in intestinalfluid at a pH greater than 7, pectin, amylose, and chitosan-basedpolymers.

It is also envisioned that a colon or tumor-specific film-coating can beused on the oral dosage form that effectively serves to increase thedelivered concentration of the composition of the present invention byselective removal of the coating by an endogenous enzyme present in thetarget tissue of the small intestines, large intestines, colon, orrectum. Alternatively, it is envisioned that tumors in the colon orrectum can be targeted by coating the dosage form with a ligand specificfor the tumor cell surface, i.e., without limitation an antibody orbinding fragment thereof.

A chewing gum is a flavored composition that is meant to deliver aflavor or other substance by chewing. The primary part of chewing gum isthe gum base, which is a non-nutritive substance. The gum base mayconsist of any one or more of the following; an elastic ingredient, aresin that acts as a binder and softener, a plasticizer, fillers, asweetener or flavoring agent, and an antioxidant. As an example, SubpartG titled “Gum, Chewing Gum Bases and Related Substances” from The Codeof Federal Regulations (Title 21, Volume 3, Subchapter B) listsingredients that may be used in gums, including arabinogalactan, naturalgum bases including chicle, chiquibul, crown gum, gutta hang kang,massaranduba balata, massaranduba chocolate, nispero, rosidinha,Venezuelan chicle, jelutong, leche capsi (sorva) perillo, leche de vaca,niger gutta, tunu, chilte, natural rubber (smoked sheet and latexsolids), synthetic substances such as butadiene-styrene rubber,isobutylene-isoprene copolymer, paraffin, petroleum wax, petroleum waxsynthetic, polyethylene, polyisobutylene, and polyvinyl acetate,plasticizing materials including glycerol ester of partially dimerizedrosin, glycerol ester of partially hydrogenated gum or wood rosin,glycerol ester of polymerized rosin, glycerol ester of gum rosin,glycerol ester of tall oil rosin, glycerol ester of wood rosin, lanolin,methyl ester of rosin partially hydrogenated, pentaerythritol ester ofpartially hydrogenated gum or wood rosin, rice bran wax, stearic acid,sodium and potassium stearates, synthetic and natural terpene resins,antioxidants including butylated hydroxyanisole, butylatedhydroxytoluene, propyl gallate, carrageenan, carrageenan withpolysorbate 80, salts of carrageenan, furcelleran, salts of furcelleran,gellan gum, and xanthan gum. Of course, compositions in addition tothose of the present invention may be included as is known in the art toprovide desirable attributes for the gum, such as enjoyment (taste,mouth-feel, and the like), breath freshening, dental care, and oralcare.

Still further, the compositions of the present invention may be providedalong with a mucoadhesive polymer excipient, for direct delivery to amucosal surface and also for transmucosal delivery into systemiccirculation. Briefly, mucoadhesive polymer excipient refers to a polymerthat has the ability to adhere to mucin. Typically, for the compositionsof the present invention a mucoadhesive acidic polymer excipient isused, which when dissolved or suspended in water results in an acidicpH. In one embodiment, the mucoadhesive polymer will provide a polymersolution or suspension having a pH of from about 1 to about 4.5. Thepolymer may be non-ionic or anionic. Mucoadhesive polymer excipientswill typically be selected which also control the rate of release of theactive ingredient from the gel by providing a controlled rate of polymerrehydration. The mucoadhesive polymer excipient may be present in thedosage form in one embodiment in a weight percentage between 5-50%. Inanother embodiment, the excipient is present in a weight percentagebetween 5-20%. In yet another embodiment, the excipient is present in aweight percentage between 5-10%. As non-limiting examples, themucoadhesive polymer excipient may be selected from one or more of thefollowing polymers: polyacrylic acid, crosslinked polyacrylic acid,polyvinyl pyrrolidone, cross-linked polyvinyl pyrrolidone,polymethacrylic acid, polymethacrylic-acid co-polymers, carboxymethylcellulose, cellulose or derivatives thereof, or alginate.

Of course, additional substances may be included in the formulations ofthe present invention to provide additional desired properties, such assweeteners or preservatives. Sweeteners may be of natural original orsynthetic, and may include, but are not limited to, fructose, glucose,glycerol, lactitol, maltitol, maltose, sorbitol, xylitol, saccharin,aspartame, cyclamate, sucralose, or acesuflame potassium, or mixturesthereof. Preservatives are natural or synthetic chemicals that are addedto hinder spoilage, whether caused by microbial growth, or unwantedchemical changes such as, but not limited to, hydrolysis or oxidation.Suitable preservatives may include, but are not limited to, benzoicacid, sorbic acid, benzyl alcohol, benzethonium chloride, butyl paraben,cetrimide, chlorobutanol, cresol, ethyl paraben, phenol, phenoxyethanol,propylene glycol, sodium benzoate, thimersol, methyl paraben, propylparaben, or mixtures thereof.

Still yet further, the present invention provides a method of treatinginflammation, oxidative damage, or cancer, comprising administering to amammal such as a human in need thereof an effective amount of acomposition as described above. The inflammation may be associated withcancer, inflammatory bowel disease, gastroesophageal reflux disease,diarrhea, radiation-induced enteritis, chemotherapy-induced enteritis,Crohn's disease, irritable bowel syndrome, diverticulitis, ulcer,colitis, viral infection, bacterial infection, parasitic infection, andcombinations thereof. The inflammation may be partially mediated by therelease in the body of the mammal of at least one cytokine including,but not limited to, an interleukin such as IL-1, IL-6, or IL-12, orTNF-α, and IFN-α. The oxidative damage may be mediated at leastpartially by release of a free radical in the body of the mammal. Thecancer may be a skin cancer, an oral cancer, a cancer of the eye, acancer of a mucosal surface such as the vagina, the nose, and therectum, and combinations thereof. As described above, the compositionmay be formulated for administration by at least one of orally,topically, and by injection.

In yet still another aspect, the present invention provides compositionsfor treating inflammation, oxidative damage, or cancer in a mammal inneed thereof, comprising a therapeutically effective amount of acomposition as described above in a pharmaceutically acceptable vehicle.The composition may further comprise at least one additional anti-canceragent, at least one additional anti-oxidant, at least one additionalanti-inflammatory agent, and combinations thereof. The compositions maybe formulated for administration by at least one of orally, topically,and by injection as described above.

The examples provided herein are presented in support of and to furtherillustrate the invention as described above and in the accompanyingdrawings, but are not to be considered as limited thereto. Citations ofliterature herein are incorporated into this disclosure by reference intheir entirety unless otherwise indicated. Generally,2,2-Azinobis(3-ethylbenzothiazoline-6-sulfonicacid)diammonium salt(ABTS), Trolox (6-hydroxy-2,5,7,8-tetramethychroman-2-carboxylic acid),potassium persulfate, formic acid (ACS, >96%),3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT),Folin and Ciocalteau phenol reagent and gallic acid (98% purity) werepurchased from Sigma (St. Louis, Mo.). Hydrogen chloride ˜1.25 M inethanol was purchased from Fluka (St. Gallen, Switzerland). USP gradeEthanol (Absolute—200 proof) was purchased from AAPER Alcohol andChemical Co. (Shelbyville, Ky.). HPLC grade acetonitrile was purchasedfrom Fisher Scientific (Fair Lawn, N.J.).

Example 1

A blackberry puree was prepared by pulping whole blackberries. Forpurposes of this disclosure, the term pulping refers to the process ofremoving the seeds and skin from whole fruit. However, it will beunderstood by those skilled in the art that the skin and seeds alsocontain many biologically useful substances and so it is envisioned thatthe skin and seeds may be retained for later use, or alternatively maybe kept with the fruit pulp rather than separated.

Blackberries were picked from the vine and placed in containers fortransporting to the picking station for inspection. The berries were notwashed in order to prevent deterioration of the whole berry by surfacewater. The seeds and skin of the berries were removed using aLangsenkamp type 161 Colossal Pulper having two agitator arms withbrushes with a stainless steel chamber, and stainless steel catch panwith two outlets (one threaded and one with a sanitary fitting) with a10-horse power, 3 phase, 60 cycle, 230/460 volt meter. Wholeblackberries were passed through the Lagsenkamp Pulper at a rate of50-75 gallons/minute to produce a homogenous blackberry puree free ofskin and seeds. The blackberry puree was stored frozen at −20° C.

Example 2

A VirTis Model AD2 Lyophilizer was used to freeze-dry a blackberrypuree, prepared as described in Example 1, to produce a free-flowingpowder. The cycle used a freezer temperature of −40° C., a condensertemperature of −50° C., and vacuum of 200 mTorr. Approximately 10 g ofblackberry puree in a glass lyophilization vial was freeze-dried. Thefollowing drying steps were used to prepare a purple cake offreeze-dried blackberry puree:

TABLE 1 Example Freeze-Drying Cycle for Blackberry Puree to ProduceActive Pharmaceutical Powder Drying Temp Time Vacuum Steps (° C.) (min)(mTorr) 1 −35 10 200 2 −30 5 200 3 −25 5 200 4 −20 1200 200 5 −15 840200 6 −10 10 200 7 −5 10 200 8 0 10 200 9 5 120 200 10 10 300 200 11 1510 200 12 20 180 200 Post Heating 25 90 200The freeze-dried blackberry puree was collected from the glasslyophilization vial and milled using a mortar and pestle to produce afree-flowing reddish-purple powder.

Example 3

A Hull lyophilizer was employed to create a second embodiment of a drypowder of the blackberry puree. A partially frozen blackberry puree(Example 1) (5204 g) was placed into the lyophilizer on metal pans inlarge chunks. The following lyophilization cycle shown in Table 2 wasused to dry the blackberry puree:

TABLE 2 Lyophilization Cycle Used to Produce Freeze-Dried BlackberryPowder Steps  1. Freeze to −40° C.  2. Raise temp to −35° C.  3. Once at−35° C., hold for 3 hr  4. Set vacuum to less 150 microns  5. Raise tempto −10° C.  6. Once at −10° C., hold for 3 hr  7. Maintain vacuum ofless than 150 microns  8. Raise temp to 0° C.  9. Once at 0° C., holdfor 3 hr 10. Raise Temp to 5° C. 11. Once at 5° C., hold for 2.5 hr 12.Raise temp to 10° C. 13. Once at 10° C., hold for 2.4 hr 14. Raise tempto 20° C. 15. Once at 20° C., hold for 2.5 hr 16. Break vacuum

The resulting blackberry powder weighed 559 g and was 10.7% w/w of theinitial blackberry puree that was lyophilized. Next, 100 g of thefreeze-dried blackberry powder was treated under sonication with 500 mLethanol containing 0.01% HCl for 30 min to produce a blackberry extracthaving a pH in the range of 1.9-2.0. The suspension was filtered and thesupernatant was collected in a flask. The supernatant was dried at 40°C. for 7 hr using a rotary evaporator. The recovered blackberry extractweighed 19.12 g or 19.12% w/w of the original 100 g dried blackberrypowder.

Example 4

Five hundred milligram portions of blackberry powder prepared asdescribed in Example 2 were evaluated for rate of water sorption. Threevials were held at 25° C. and three vials were held at 4° C. Thepercentage weight increase over time due to water sorption was recordedfor a period of 45 hr. As shown in FIG. 1, blackberry powder stored at25° C. increased in weight by up to 10% by 30 hr, and then reached aplateau. In contrast, blackberry powder stored at 4° C. increased inweight by less than 1.7% and reached a plateau after only 5 hr.

Example 5

Two different blackberry extracts were obtained by subjecting 2.5 gblackberry powder (Example 2) three times with either; sample A) 20 mLof ethanol/0.1% HCl or, sample B) 20 mL of ethanol/0.01% HCl undersonication. The three samples for each extraction condition werecollected and centrifuged for 10 min at 12,000 g. The supernatant wasthen filtered to collect dark red solution. Ethanol was then removed at40° C. using a rotary evaporator to obtain a dark red, viscous liquid.Ten (10) mL of water was added to sample A to produce a dark-redsuspension having a pH of 1.2. Ten (10) mL of water was added to sampleB to produce a dark-red suspension having a pH of 1.9. Both sample A andsample B were centrifuged for 10 min at 12,000 g and a final dark-redsolution referred to berry extracts. As will be discussed in greaterdetail below, the resulting compositions are enriched for stabilizedanthocyanins.

Example 6

An aliquot of freeze-dried blackberry powder (2.5 g) prepared asdescribed in Example 2 was treated under sonication for 30 min with 60mL of ethanol containing 0.01% HCl. The supernatants were collectedafter filtration and dried by rotary evaporation at 40° C. The driedextract was dissolved in 10 mL of deionized water and filtered through a1.0 μm nylon syringe filter. The blackberry extract had a pH of 1.9. Theextract was either frozen at −20° C., or lyophilized to produce a driedblackberry extract (DBE) and then stored as a powder at −20° C. Theaverage yield of DBE was 470.44±9.27 mg per gram of blackberry powder.

The blackberry extract was characterized for: i) monomeric anthocyaninsand polymeric color measurement, ii) Total Phenolic Measurement, iii)Trolox Equivalent Antioxidant Capacity (TEAC) Assay, and iv) anthocyanincomposition. Results of characterization for i, ii, and iii are shown inTable 3 below. It is important to note that the values for these severalparameters are likely influenced by the extraction efficiency of theberry extract. Thus, it may be appreciated by those skilled in the artthat a different extraction protocol may result in a berry extract thathas different composition and features.

TABLE 3 Composition and Characterization of Dried Blackberry Extract(DBE) Total Total TAC^(c) Anthocyanins^(a) Phenolics^(b) Polymeric (μMTE^(d)/ (mg/g DBE) (mg/g DBE) color (%) g DBE) Blackberry 6.80 + 0.3117.32 ± 0.74 2.2 ± 1.0 66.98 ± 1.27 extract (n = 3) ^(a)Totalanthocyanins were expressed as cyanidin-3-glucoside equivalent.^(b)Total phenolics were expressed as gallic acid equivalent. ^(c)TAC:total antioxidant capacity measured by Trolox-equivalent antioxidantcapacity assay. ^(d)TE: Trolox equivalent.

i) Monomeric Anthocyanins and Polymeric Color Measurement. Monomericanthocyanin content was determined by the pH-differential method ofGiusti and Wrolstad (2001). Briefly, samples were diluted 1:100 v/v in a25 mM potassium chloride buffer (pH 1.0) and a 0.4 M sodium acetatebuffer (pH 4.5), then read against a blank at 510 nm and 700 nm with 1cm path length disposable cuvettes. Total anthocyanin content wascalculated using the equations below.

A=(A ₅₁₀ −A ₇₀₀) pH 1.0−(A ₅₁₀ −A ₇₀₀) pH 4.5  Equation (1)

C (mg/L)=(A×MW×DF×1000)/(ε×1)  Equation (2)

-   -   Where:        -   A is the absorbance of the diluted anthocyanin sample;        -   C is the anthocyanin concentration (mg/L);        -   MW is the molecular weight (449.2 for cyanidin-3-glucoside);        -   DF is the dilution factor; and        -   ε is the molar absorptivity (26900 for            cyanidin-3-glucoside).            Color density and polymeric color were calculated using            absorption at 420, 510 and 700 nm with and without bisulfite            treatment. The percentage of polymeric color was determined            by the ratio of polymerized color to color density.

ii) Total Phenolic Measurement. Total phenolic content in the berryextract was estimated based on the method of Singleton and Rossi (1965)using gallic acid as a standard. Twenty (20) μL of diluted samples wasadded to 1.58 mL distilled water along with 100 μL of a 2NForlin-Ciocalteu phenol reagent. All solutions were mixed thoroughly andthen allowed to sit at room temperature for 1 min. Three hundred (300)μl saturated sodium carbonate solution (200 g/L) was then added to eachsample. The absorbance was measured at 765 nm with a Beckman UV-visiblespectrophotometer after incubation for 2 hr at room temperature. Totalphenolics were determined based on the standard curve generated with0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL of gallic acid.

iii) Trolox Equivalent Antioxidant Capacity (TEAC) Assay. TEAC assay forthe berry extract was carried out using a Beckman DU640Bspectrophotometer following procedures described by Re et al., (1999).ABTS•+ was produced by reacting 7 mM ABTS with 2.5 mM potassiumpersulfate for 16 h in the dark at room temperature. The ABTS•+ solutionwas diluted with ethanol to an absorbance of 0.70 (±0.02) at 734 nm andequilibrated at 30° C. Twenty (20) μl of the berry extract samples wereadded to 980 n1 of diluted ABTS•+ solution, such that each final sampleproduced between 20-80% inhibition of the blank absorbance. Theabsorbance readings were taken continuously every 6 s for 6 min at 734nm at 30° C. Trolox standards in ethanol with final concentrationranging from 0 to 16.8 μM were prepared and assayed under the sameconditions. The total antioxidant capacity of the berry extract wascalculated and expressed as μmol Trolox equivalent (TE) per gram ofdried blackberry extract (DBE). The total antioxidant capacity (TAC) ofthe berry extract was determined using an improved TEAC assay (Re etal., 1999), which assessed the capacity of a compound or sample toscavenge ABTS•+ in terms of Trolox equivalent. The average TAC value ofthe berry extract was 66.98±1.27 mmol TE per gram of DBE (see Table 3).

iv) Anthocyanin Composition. HPLC-UV-MS analysis was performed using aX-Bridge™ C18 column (250 mm×4.6 mm, 5 μm) (Waters) equipped with anX-Bridge™ C18 guard column with a Waters 2690 separation module equippedwith a 996 photodiode array detector, and coupled on-line with a WatersMicromass ZMD 4000 Mass Spectrometer. The mobile phase consisted of 10%formic acid (A) and 100% acetonitrile (B). The elution conditions wereas follows: 0-45 min, linear gradient from 1 to 16% B (v/v); 46-50 min,linear gradient from 16% to 100% B; 51-60 min, 100% B; post-time 5 minwith 1% B; flow rate 1 mL/min. The UV-visible detection wavelength was524 nm and the injection volume was 50 μL of the berry extract. The MSinstrument was operated at the following settings: ESP+ mode; capillaryvoltage, 3.0 kV; cone voltage, 35 V; desolvation temperature, 300° C.;source temperature, 100° C.; scan range, 100-1000 m/z. The HPLC profileof the berry extract showed six major peaks as identified as 1-6 (FIG.2). Peak identification was carried out based on the molecular weightand structural information obtained from their MS spectra, in additionto their retention times from HPLC-UV-vis spectra.

It is notable that other small peaks as shown in the chromatogram inFIG. 2 were not identifiable by MS and their identity remains unknown.Cyanidin-3-glucoside (peak 1) was the main component (71.0%) in theberry extract with the respective parent and daughter ion pairs (m/z449/287). The other three major peaks (peak 4, 12.4%; peak 5, 3.5%; peak6, 11.6%) revealed the m/z values of 419/287, 535/287 and 593/287 whichwere identified as cyanidin-3-xyloside, cyanidin-3-malonylglucoside andcyanidin-3-dioxalylglucoside respectively, in accordance with datapreviously reported by Stintzing et al. (2002). Cyanidin-3-arabinoside(peak 2) was also identified, and was in agreement of the initial reportby Dugo et al. (2001) using blackberry extracts. Another small peak(peak 3), with the respective parent and daughter ion pairs (m/z435/303), was detected in the blackberry extract and is being reportedfor the first time. Based on its retention time, this compound has beententatively identified as delphinidin-3-xyloside.

Example 7

An HPLC assay was used to quantify the anthocyanin content and stabilityin berry samples. The standard chromatogram gave two peaks correspondingto: peak 1=cyanidin-3-glucoside and cyanidin-3-sambubioside; and peak2=cyanidin-3-rutinoside and cyanidin-3-(2G-xylosylrutinoside). Astandard curve showed excellent linearity over a range of from 50 to2000 μg/mL with R²>0.999 for both peak 1 and peak 2. FIG. 3A depicts thestability of peak 1 and peak 2 after one day at 4° C. in formulationsmade from pH 4.5 to 7.5. FIG. 3B depicts the stability of peak 1 andpeak 2 after 28 days at 4° C. in formulations made at pH 3.5 or pH 4.0.

Freeze-dried berries (prepared as described in Example 2) wereformulated at a final concentration of 10% w/w in mixtures havingdifferent pHs from pH 7.5, 6.5, 5.5, 4.5, 4.0, and 3.5 and stored at 4°C. for 1 day. As shown in FIG. 3A, peak 1 and peak 2 showed much greaterstability at lower pH values after 1 day. The results of a second studyare shown in FIG. 3B. In this study, the berries were formulated at pH3.5 and 4. Formulation of the berries at pH 3.5 provided excellentstability for peak 1 (96.0±0.8%) and peak 2 (100.2±0.4%) over 1 month at4° C.

Example 8

Blackberry extract (19.12 g, see Example 3) was dissolved in 500 mLwater to produce a stock solution of 38.24 mg extract/mL. Totalanthocyanin content of the 19.12 g blackberry extract was found to be181.3 mg/L by the pH differential method. This corresponded to a totalanthocyanin content of 90.6 mg from the 19.12 g of blackberry extract(0.5% w/w anthocyanins).

Two (2) mL of the blackberry extract, corresponding to 76.48 mg extractwas mixed with 8 mL of 10% mannitol and lyophilized to produce a driedberry extract with mannitol. The final mannitol concentration in thesolution was 8% and the final pH ranged from 2.5 to 2.6. In addition,blackberry extract alone (without mannitol) was lyophilized to produce adried berry extract. The lyophilization procedure for both the driedberry extract with mannitol and dried berry extract is shown in Table 4.

TABLE 4 Lyophilization Cycle Used to Prepare Dried Berry Extract andDried Berry Extract with Mannitol Steps  1. Freeze to −40° C.  2. Raisetemp to −35° C.  3. Once at −35° C., hold for 3 hr  4. Set vacuum toless 150 microns  5. Raise temp to −10° C.  6. Once at −10° C., hold for15 hr  7. Maintain vacuum of less than 150 microns  8. Raise temp to 0°C.  9. Once at 0° C., hold for 3 hr 10. Raise Temp to 5° C. 11. Once at5° C., hold for 10 hr 12. Raise temp to 5° C. 13. Once at 10° C., holdfor 3.4 hr 14. Raise temp to 20° C. 15. Once at 20° C., hold for 2.5 hr16. Break vacuum

To observe the stability of the blackberry extract in solution alone,and in the presence of 8% mannitol, the extract was stored at 4° C. and25° C. for 1 month. As shown in Table 5 below, at both temperatures, thepresence of 8% mannitol in solution was able to stabilize the retentionof four different anthocyanins in solution designated by Peak 1(Cn-3-glucoside), Peak 2 (Cn-3-arabinoside), Peak 3 (Cn-3-xyloside), andPeak 4 (Cn-3-malonyl-glucoside).

TABLE 5 Retention of Anthocyanins in Berry Extract Solution and BerryExtract with 8% Mannitol Solution Over 1 Month Peak 1 Peak 2 Peak 3 Peak4 % Retention of Anthocyanin Peak Area after Storage at 4° C. for 1month Berry Extract  89.7 ± 0.3  86.2 ± 13.2  90.7 ± 3.1 88.0 ± 6.7 inSolution Berry Extract 106.0 ± 1.0 138.0 ± 5.9  109.5 ± 4.9 115.6 ± 10.7with Mannitol % Retention of Anthocyanin Peak Area after Storage at 25°C. for 1 month Berry Extract  79.2 ± 0.2  75.5 ± 10.8  81.3 ± 2.4 69.6 ±3.4 in Solution Berry Extract 101.9 ± 0.6 96.7 ± 3.4 102.0 ± 4.6  77.2 ±24.3 with Mannitol

To observe the stability of blackberry extract in solution stored atfour different temperatures or conditions, blackberry extract solutionwas stored frozen at −80° C. as a control, at 4° C., at 25° C. in thedark, and at 25° C. with light for 90 days. As shown in FIG. 4, theconcentration of cyanidin-3-glucoside remained unchanged over 90 dayswhen the blackberry extract solution was frozen at −80° C. When theblackberry extract solution was stored at 4° C. over 90 days, there wasabout a 10% loss in the concentration of cyanidin-3-glucoside. Incontrast, there was substantial loss of cyanidin-3-glucoside over 90days when the blackberry extract solution was stored at 25° C.,indicating that the anthocyanin was less stable as the temperatureincreased.

To further confirm the stability of dried blackberry extracts storedunder various conditions and at various temperatures, freeze-driedblackberry powder was stored at −20° C. as a control, dried berryextract was stored at 4° C. and 25° C., and dried berry extract withmannitol stored at 4° C. and 25° C. The stability time points collectedwere at time 0, 2 weeks, 4 weeks, and 8 weeks.

Sample quantitation was performed by comparing the area of eachstability data point to the area for the Time 0 point for each type ofsample (freeze-dried blackberry powder, dried extract, or dried extractwith mannitol). Equation 3 was used to calculate the concentration(mg/mL) of the sample using the actual weight of sample.

$\begin{matrix}{C = {\begin{matrix}{Sample} \\{concentration}\end{matrix}\mspace{14mu} = \frac{{Actual}\mspace{14mu} {Weight}\mspace{14mu} {of}\mspace{14mu} {Sample}}{{Volume}\mspace{14mu} {of}\mspace{14mu} {Dilution}}}} & {{Equation}\mspace{14mu} (3)}\end{matrix}$

The percent of each sample peak area relative to the peak area at theTime 0 point was calculated using Equation 4.

$\begin{matrix}{\begin{matrix}{\% \mspace{14mu} {Peak}} \\{Area}\end{matrix} = {\frac{\begin{matrix}{{Peak}\mspace{14mu} {Area}} \\{{at}\mspace{14mu} {Timepoint} \times C_{{Time}\; 0}}\end{matrix}}{{Peak}\mspace{14mu} {Area}\mspace{14mu} {at}\mspace{14mu} {Time}\mspace{14mu} 0\mspace{14mu} C_{Timepoint}} \times 100}} & {{Equation}\mspace{14mu} (4)}\end{matrix}$

As shown in FIG. 5A, the Cn-3-glucoside in the freeze-dried blackberrypowder stored at −20° C. was stable for at least eight weeks. Likewise,Cn-3-glucoside remained stable in both the dried blackberry extract andthe dried blackberry extract with mannitol stored for 8 weeks at 4° C.Cn-3-glucoside was continuously lost in the dried blackberry extractover 8 week storage at 25° C. After 8 weeks, the retained Cn-3-glucosidewas only about 65% of the original. In contract, Cn-3-glucoside remainedstable in the dried blackberry extract with mannitol stored for 8 weeksat 25° C. Surprisingly, this suggested that mannitol had a stabilizingeffect on the dried blackberry extract and in particular Cn-3-glucoside.As shown in FIG. 5B, this effect was also observed for at least threeother anthocyanins. FIG. 5B shows the stability of Cn-3-glucoside,Cn-3-arabinoside, Cn-3-xyloside, and Cn-3-malonyl-glucoside over 8 weeksat 25° C. stored as either a dried blackberry extract or driedblackberry extract lyophilized in 8% mannitol.

Example 9

A blackberry extract was obtained by extracting 2.5 g blackberry powder(prepared as described in Example 2) three times with ethanol/0.01% HCl(see Example 4). The three extracts were collected and the ethanol wasremoved by rotoevaporation. Ten (10) mL of water was then used torehydrate the reddish purple extract. One (1) mL of the blackberryextract was then added to 4 mL of mannitol at a concentration of 10%,5%, 2%, 1%, or 0%. The blackberry extract/mannitol samples were thenlyophilized as described in Example 2. A pink free-flowing powder wasonly obtained with final mannitol concentration of >8%. The pH of theblackberry extract/mannitol mixture was in the range of pH 2.5 to 2.6.Freeze-drying of the blackberry extract in final mannitol concentrationsless than 2% resulted in a sticky gelatin-like product.

Example 10

A vanishing lotion base having the composition presented in Table 6 wasprepared by heating a water phase to 70° C. An oil phase was heated toabout 60° C. and added to the stirring water phase. The resultingmixture was stirred at 70° C. at 1,000 rpm for 30 minutes to produce ahomogenous oil-in-water emulsion. After 30 minutes the oil-in-wateremulsion was allowed to mix at room temperature at 1,000 rpm for 2hours. The lotion was q.s. to 100% and a pH of 3.1. The lotion was thenallowed to sit at room temperature overnight to cure. To 18 g of thevanishing lotion base, 2 g of blackberry powder (Example 2) was added toproduce a final 10% blackberry lotion. The lotion had a purple colorthat when applied to human skin and rubbed, vanished within 10-15seconds.

TABLE 6 Composition of Vanishing Lotion Base Used to Prepare a 10%Blackberry Lotion Lotion Formula Ingredients % (w/w) Water 83.39Propylene Glycol 3.33 Sorbitol, 70% 2.22 Sorbic Acid 0.22 ButylatedHydroxytoluene 0.11 Simethicone 0.11 Sub-total Water Phase 89.38Petrolatum 3.89 Cetostearyl Alcohol 3.06 Brij 58 2.78 GlycerylMonostearate 0.22 PEG 400 Monostearate 0.67 Sub-total Oil Phase 10.62Total 100

Example 11

The composition described in Example 10 was prepared, except that theblackberry extract described in Example 3 was used. The lotion wasprepared identically as described in Example 10 except that to 18 g ofthe vanishing lotion base (Table 6), 2 g of dried blackberry extract(Example 3) was added to produce a final 10% blackberry extract lotion.The lotion had a purple color that when applied to human skin andrubbed, vanished within 10-15 seconds.

Example 12

A 1 kg vanishing cream base was prepared using the formula set forth inTable 7.

TABLE 7 Formula for Preparing Vanishing Cream Base Calculated weight (g)Ingredients % w/w for 1 kg batch Water for injection 79.8 798 PropyleneGlycol 3.0 30 Sorbitol, 70% 2.0 20 Sorbic Acid 0.2 2 ButylatedHydroxytoluene 0.1 1 Simethicone 0.1 1 Sub-total water phase 85.2 852Petrolatum, white 5.6 56 Cetostearyl alcohol 4.4 44 Brij 58 4.0 40Glyceryl monostearate 0.2 2 PEG-400 monostearate 0.6 6 Sub-total oilphase 14.8 148 TOTAL 100 1000

The cream was prepared by first preparing the water phase. Water (798 g)was added to the mixing vessel followed by 30 g propylene glycol. Thiswax mixed until homogeneous. Next 20 g of 70% sorbitol was added, andthen 2 g sorbic acid. This was mixed until homogeneous and then headedto 60-70° C. Butylated hydroxytoluene (1 g) was then added and mixedfollowed by 1 g simethicone. The water phase was mixed until homogenousat 60-70° C. To prepare the oil phase, the following materials wereadded to a separate vessel; 56 g white petrolatum, 44 g of cetostearylalcohol, 40 g of Brij 58, 2 g of glyceryl monostearate, 6 g ofpolyethylene glycol 400 monostearate. After the addition, the oil phasewas heated at 60-70° C. until melted to produce a homogenous phase.Next, the melted oil phase was slowly added to the oil phase and mixedto obtain a homogenous mixture between 60-70° C. The mixture was stirredfor an additional 30 min, and then allowed to cool while stirring to30-35° C. The net weight was adjusted to 1000 g with water and thenmixed to produce a homogeneous white placebo cream, pH 3.2. To thiscream, freeze-dried blackberry powder (Example 2) was added directly tothe placebo cream to produce 1%, 5%, and 10% blackberry cream.Separately, freeze-dried blackberry extract prepared as described inExample 3 was added to produce 1%, 5%, and 10% blackberry extract cream.The pH of all creams was adjusted to pH 3.5 by adding 2N potassiumhydroxide. Creams containing either freeze-dried blackberry powder orblackberry extract were pink-red in color depending on the finalconcentration. All creams applied to human skin and rubbed vanished intothe skin with 10-15 seconds after continuous rubbing.

Example 13

An enteric-coated blackberry powder capsule was prepared. Empty hardgelatin capsules (#3 Coni-Snap white opaque, Capsugel Lot #589552)having a mean capsule weight of 46.53±0.61 mg were filled with 120 mg ofactive blackberry powder (Example 2). Fifteen capsules each wereenteric-coated with either Coating Solution A or Coating Solution B:

Coating Solution A: 12.50 g EUDRAGIT L-100 and 6.25 g triethyl citratedissolved in 81.25 g of acetone: isopropyl alcohol (60:40 v/v).Coating Solution B: 12.50 g EUDRAGIT S-100 and 6.25 g triethyl citratedissolved in 81.25 g of acetone: isopropyl alcohol (60:40 v/v).

As is known in the art, EUDRAGIT L-100 is soluble above pH 6, whereasEUDRAGIT S-100 is soluble above pH 7. For each coating solution (A andB), 15 individual capsules were coated by taking the capsule fullyclosed from one end and dipping it in the solution for 2 seconds. About60% of the capsule was submerged. Residual coating solution was removedby gently touching tip of capsule to a cloth. The solution wasevaporated in a stream of airflow from the hood. After 2 hours, thecapsule was inverted and the same procedure was repeated. After 2 hoursthe 15 dry coated capsules were individually weighed and the means werecalculated for each coating solution. The average weight of the coatingfilm for each coating solution was calculated, subtracting the averageweight of an uncoated capsule from the average weight of thecorresponding coated capsule. The average coating weight for capsulescoated with coating Solution A and Solution B was 5.53 mg and 4.92 mg,respectively.

A modified version of the disintegration procedure from USP 25 (<701>)was followed using three individual enteric-coated capsules for eachcoating solution and for each of four disintegration fluids (pH 1, pH4.5, pH 6 and pH 7.4). One capsule was placed in each of three tubes ofthe basket in the disintegration apparatus. One liter of each buffermaintained at 37° C.±2° C. during the test was used as the immersionfluid and the disintegration time of capsules was recorded. The endpoint of the disintegration process was taken to be when the capsule hadopened completely and disintegrated except for small fragments of thecapsule shell. The results are presented in Table 8.

TABLE 8 Disintegration Time of Blackberry Capsules Enteric-Coated withEUDRAGIT L-100 or EUDRAGIT S-100 as a Function of pH pH pH = 1.0 pH =4.5 pH = 6.0 pH = 7.4 Buffer (10 mM (10 mM (10 mM Type of (0.1MCitric-Citrate Phosphate Phosphate Capsule HCl) Buffer) Buffer) Buffer)Uncoated Capsule <5 min <5 min  <5 min  <3 min 1 Capsule <5 min <5 min <5 min  <3 min Capsule <5 min <5 min  <5 min  <3 min 3 EUDRAGITCapsule >1 hour >1 hour <25 min  <7 min L-100 1 Coated Capsule >1hour >1 hour <25 min  <7 min Capsule >1 hour >1 hour <25 min  <7 min 3EUDRAGIT Capsule >1 hour >1 hour  >1 hour <28 min S-100 1 CoatedCapsule >1 hour >1 hour  >1 hour <28 min Capsule >1 hour >1 hour  >1hour <28 min 3 Note: For “>1 hour”, samples were removed from the vesselintact at 1 hour

Next, a simulated dissolution study was completed at 37° C.±2° C. usingthe blackberry capsule coated with EUDRAGIT L-100 (as noted above, apolymer coating that dissolves at a pH greater than 6). The results aresummarized in FIG. 6. Two capsules were placed in medium having pH 1 for1 hour at 37° C.±2° C., and then transferred intact to medium of eitherpH 6 or pH 7.4. EUDRAGIT L-100 coated capsules disintegrated veryrapidly at pH 7.4 and released their contents. However, the capsulesdisintegrated more slowly at pH 6 and had released only about 50% oftheir contents within the first 60 minutes at pH 6.

Example 14

Coni-Snap (#2) white opaque capsules from Capsugel were used to prepareenteric-coated capsules comprising blackberry extract with mannitolprepared as described in Example 8. An enteric-coating solution was usedto coat the capsules. The coating solution was made by dissolvingEudragit L-100 (12.54 g) in 81.26 g coating solvent containing 6.14 gtriethyl citrate. The coating solvent was acetone:isopropyl alcohol(60:40 v/v). Empty uncoated capsules (average weight of 58.6±0.9 mg)were hand-filled with dried blackberry extract with mannitol powder. Theaverage weight of filled capsules was 134.4±15.3 mg. Filled capsuleswere then enteric-coated with Eudragit L-100 using a Torpacenteric-capsule coater. The coated capsules were allowed to dry.

A two-phase pH dissolution study was performed by first exposing acoated capsule to 30 mL 0.1N HCl, pH 1 at 37° C. for 30 min whilestirring at 50 rpm. Two mL samples were removed at 15 min and 30 min toassay for anthocyanin release. After 30 min, the capsule was transferredto 30 mL PBS, pH 7.4 buffer at 37° C. stirring at 50 rpm. Two (2) mL ofsample was removed from the PBS after 10, 30, 45 and 60 min. Twenty (20)μl of formic acid (1% final formic acid concentration) was added to eachsample to acidify. The volume lost in the vessel each time was replacedwith 2 mL of PBS. The results as shown in FIG. 7 demonstrate thatanthocyanins were not released from the intact capsule at pH 1 for 30min. However, after transfer to pH 7.4, the capsules underwentdisintegration and anthocyanins were released. Plotted in FIG. 7 is theaverage anthocyanin released for three capsules.

Example 15

A chewing gum base (see Table 9) was prepared by preheating a gum baseto 150° F. and a Sigma blade mixer to 120° F. About 40% of Sorbogem712Crystalline Sorbitol was added to the warmed mixer. The preheated gumbase was added to the mixer, followed by an additional 40% of theSorbogem 712 Crystalline Sorbitol. The liquids were then blended exceptfor the aspartame and spearmint flavor. The liquids were then added tothe preheated mixer and the mixer was turned on. The remaining 20% ofthe Sorbogem712 Crystalline Sorbitol was added.

The entire blend was mixed for 3 minutes and then the aspartame wasadded. The gum was then mixed an additional 5 minutes and then thespearmint flavor was added. The gum was then mixed for an additional 3minutes and then blackberry powder (Example 2) was added so that thefinal blackberry powder was 5% of the total weight. The blackberry gumwas mixed an additional 3 minutes and the gum was removed form themixer. The blackberry gum was sheeted, scored, and cut to predeterminedportion sizes.

TABLE 9 Composition of Gum Base Used to Prepare a 10% Blackberry ChewingGum Gum Base Ingredients % (w/w) SORBOJEM 712 Crystalline Sorbitol 49.6PALOGA Gum Base 25.7 Mannitol 10.0 MALTISWEET 3145 Maltitol Syrup 9.7Glycerin 3.9 Spearmint Flavor 1.0 Aspartame 0.1 Total 100

Example 16

A second embodiment of a blackberry chewing gum was prepared. Silicondioxide, magnesium stearate, sorbitol, and mannitol were obtained fromSpectrum. Pharmagum C was purchased from SPI Polyols. Carmine 52% PurpleType Color and Natural Blackberry Flavor were obtained from WildFlavors. Sugartab was purchased from JRS Pharma.

Compressible chewing gum blends were prepared as shown in Table 10 todetermine the needed compression force to form a suitable tablet. Theformulation powder was prepared using the target weight of 2000 mg perchewing gum tablet. The following procedures were used to prepare theformulation mixtures. Pharmagum C was placed in the V-blender(Patterson-Kelly Co.) along with the liquid flavor and the mix wasallowed to blend for 2 min. To this blend was the first addition ofsilicon dioxide to adsorb any residual liquid and the mix was allowed toblend for 15 min. A sieve was used to break up any clumps that may haveformed during this addition. Dried berry extract with mannitol (Example7) was removed from the vials, weighed and placed in the V-blender; themix was allowed to blend for 5 min. Sorbitol was then added and the mixwas allowed to blend for 5 min. Powdered color was added if needed andthe mix was allowed to blend for 5 min. Magnesium stearate was thenadded and the mix was allowed to blend for 5 min. Finally, the secondaddition of silicon dioxide was added and the mix was allowed to blendfor 5 min.

Two thousand (2000) mg of a gum formulation were placed in the punch anddie set (0.6500″, square arc, punch/die, Natoli Engineering). Chewinggum was compressed using a Carver Carver Hydraulic Tablet press (ModelC) at compression forces of 2500, 3000, 3500, and 10,000 pounds ofpressure. Formulation #16 pressed at 10,000 pounds of pressure was foundto be the most desirable chewing gum formulation in terms of hardnessand sweetness.

TABLE 10 Composition (%) of Excipients in the Blackberry Flavored GumFormulations Berry Liquid Silicon Powdered Extract Magnesium PharmagumFlavor Dioxide Sorbitol Sugartab Flavor w/Mannitol Color StearateMannitol 1 75.0 2.0 2.0 10.0 0.0 8.0 0.0 0.5 0.5 2.0 2 71.0 4.0 4.0 10.00.0 8.0 0.0 0.5 0.5 2.0 3 67.0 6.0 6.0 10.0 0.0 8.0 0.0 0.5 0.5 2.0 463.0 8.0 8.0 10.0 0.0 8.0 0.0 0.5 0.5 2.0 5 71.0 4.0 4.0 12.0 0.0 6.00.0 0.5 0.5 2.0 6 73.0 4.0 2.0 12.0 0.0 6.0 0.0 0.5 0,5 2.0 7 69.0 6.04.0 0.0 12.0 6.0 0.0 0.5 0.5 2.0 8 73.0 4.0 2.0 0.0 12.0 6.0 0.0 0.5 0.52.0 9 70.0 5.0 3.0 0.0 8.0 0.0 0.0 0.5 1.0 12.5 10 69.0 5.0 3.0 0.0 8.00.0 0.0 0.5 2.0 12.5 11 69.0 6.0 3.0 0.0 8.0 0.0 12.5 0.5 1.0 0.0 1268.0 7.0 3.0 0.0 7.0 0.0 12.5 0.5 1.0 0.0 13 69.0 5.0 3.0 0.0 8.0 0.012.5 0.5 2.0 0.0 14 68.0 7.0 3.0 0.0 7.0 1.0 12.5 0.5 1.0 0.0 15 69.05.0 3.0 0.0 8.0 0.0 12.5 0.5 2.0 0.0 16 69.0 5.0 3.0 0.0 8.0 0.0 12.50.5 2.0 0.0

Example 17

To evaluate health and medical benefits, and in particularanti-proliferative/anti-cancer effects, of the composition of thepresent invention, a blackberry extract was obtained as described inExample 4 by extracting 2.5 g blackberry powder (prepared as describedin Example 2) three times with 20 mL of ethanol/0.01% HCl undersonication. The three samples were collected, combined, and centrifugedfor 10 min at 12,000 g. The supernatant was then filtered to collectdark red solution. Ethanol was then removed at 40° C. using a rotaryevaporator to obtain a dark red, viscous liquid. Ten (10) mL of waterwas added to produce a dark-red suspension having a pH of 1.9. Thissample was then centrifuged for 10 min at 12,000 g to obtain a finaldark-red solution.

HT-29 human colorectal cancer cells (ATCC, HTB38) were grown in McCoy's5A medium supplemented with 10% fetal bovine serum, 100 IU/mLpenicillin, and 10 μg/mL streptomycin and maintained in a humidifiedatmosphere with 5% CO₂ at 37° C. For the cell proliferation assay, HT-29cells were seeded at a density of 1.2×10⁴ cells/well in 96-well platesand incubated under normal growth conditions overnight to allow cells toattach and proliferate. The berry extract was added in finalconcentrations ranging from 13.6 to 49.2 μg monomeric anthocyanins/mLmedium to each well. Vehicle controls were the normal media with thecorresponding pH adjusted using 2.5 N HCl solution of each treatedgroup. At the 0.5, 2, 4 hr time points, the medium was replaced withfresh medium and the cells were cultured for a total of 48 or 72 hr.Cell growth was measured using the MTT assay as previously described(Visconti et al., 1999). Briefly, an MTT stock solution (5 mg/mL) wasprepared by dissolving MTT in PBS, pH 7.4. The stock solution was addedat 1:10 v/v to the medium in each well, and plates were incubated in thedark at 37° C. for 4 hr. Next, supernatant was removed and 200 μl of0.04 N HCl in isopropanol was added to each well. After keeping in thedark at room temperature for 1 hr, plates were read at 570 nm using anELISA plate reader. Cell viability was calculated using Equation 5:

% inhibition=(ABS _(Ctrl) −AB _(St))/ABS _(Ctrl)×100%  Equation (5)

where AB_(St) is an absorbance of cells treated with the berry extractand ABS_(Ctrl) is the absorbance of corresponding vehicle control.

There are numerous reports on the growth inhibition of cancer cells invitro by various anthocyanin-containing extracts or purified anthocyaninfractions from various kinds of fruits and vegetables (Olsson et al.,2004; Seeram et al., 2004; Zhao et al., 2004; Reddy et al., 2005; Yi etal., 2005; Zhang et al., 2005). Among them, Olsson et al. showed anaverage inhibition of 53% on HT29 cells at the highest concentration ofstrawberry extracts (Olsson et al., 2006). Yi et al. found that the IC₅₀for blueberry extracts with HT29 cells ranged from 1000-3000 μg/mL (Yiet al., 2005). Commercially prepared grape, bilberry, and chokeberryanthocyanin-rich extracts (AREs) were investigated by Zhao et al. and itwas demonstrated that all of the three extracts inhibited HT29 cellgrowth, with chokeberry ARE being the most potent inhibitor (Zhao etal., 2004). In addition, Parry et al. reported similar results withblack raspberry, cranberry, and chardonnay grape seed flour extracts onthe anti-proliferative effects on HT29 cells (Parry et al., 2006).However, for all of these studies mentioned above, the HT29 cells weretreated with extracts for at least 24 hr. In addition, to our knowledge,the present study is the first to investigate the growth inhibitioneffect of an aqueous extract from blackberries on the proliferation ofHT29 cells. Moreover, in the present studies, cells were treated with areduced contact time of 0.5 to 4 hr instead of the typical 24 hr. It wasfound that the berry extract inhibited the growth of HT-29 cells in aconcentration-dependent manner. In addition, the inhibition rates ofHT-29 cells versus anthocyanin concentration were comparable in thesestudies to extracts derived from other fruits.

As shown in FIG. 8, HT-29 cells were exposed to berry extract for 2 hrand then incubated with fresh media for 48 or 72 hr. The growth of HT-29cells was inhibited by 24% to 53% (P<0.001) at concentrations rangingfrom 13.6 to 49.2 μg monomeric anthocyanins/mL medium at 48 hr At the 72hr time point, although the HT-29 cell growth inhibition was notsignificant (P=0.2) at the concentration of 13.6 μg monomericanthocyanins/mL medium, significant inhibition of 39% and 66% (P<0.001)was observed at higher concentrations of 32.8 and 49.2 μg monomericanthocyanins/mL medium, respectively. The studies by Malik et al. (2003)and Zhao et al. (2004) using anthocyanin-rich berry extracts suggestedthat the increase in inhibition with exposure time (24, 48 and 72 hr)was mainly due to growth of control cells over time as there was littleor no change in the growth of cells exposed to the berry extracts. Incontrast, in the present studies, cells were exposed to berry extractfor a fixed period of time and then removed and replaced with freshmedia for addition incubation for 48 or 72 hr. As a consequence, a trendof increasing inhibition over time was not observed in the presentstudies. For example, the percentage inhibition of 38.6% and 38.99% werenot statistically different at the 48 and 72 hr time point after twohour exposure of a concentration of 32.8 monomeric anthocyanins/mLmedium, respectively. Similar growth inhibition rates and patterns werealso observed at 48 and 72 hr time point after 0.5 hr and 4 hr exposureof the berry extract (data not shown) which may indicate that the activecomponents (including anthocyanins) that lead to inhibition by the berryextract may be rapidly taken up by the cells.

Example 18

The experiment described in Example 17 was repeated, with the exceptionthat media containing berry extract was added to the HT-29 cells afterincubation for 0.5 hr at 37° C., rather than adding berry extractdirectly to the cells. The results showed a similar inhibition rate atall concentrations as the berry extract with a concentration of 13.6 μgmonomeric anthocyanins/mL medium was added directly to cells. Sinceanthocyanins are known to be less stable at neutral pH than low pH, thisresult may suggest that there are other active components in the berryextract. However, this aspect was beyond the scope of the presentstudies and remains an active area of investigation.

There are a few studies that have been performed to elucidate themechanism behind the chemoprevention effects of anthocyanins oranthocyanin-rich extracts on cancer cells. Hibiscus (Chang et al.,2005), Lingonberry (Wang et al., 2005), and bilberry (Katsube et al.,2003) anthocyanins-rich extract were found inhibiting the growth of HL60(human leukemia cells) through the induction of apoptosis. Hou et al.showed anthocyanins inhibit tumorigenesis induced by TPA on mouse JB6(+) cells by blocking activation of the MAPK pathway (Hou et al., 2004).Several groups have reported that anthocyanins could suppress thecyclooxygenase activity, which may also play a key role oncarcinogenesis (Wang et al., 1999; Seeram et al., 2001; Hou et al.,2005). Hakimuddin et al. found that the inhibition of MCF-7 cellproliferation by a flavonoid fraction from a red wine was related to itsinhibition of calcium and calmodulin-promoted phosphodiesterase activity(Hakimuddin et al., 2004). The molecular mechanism by which the berryextract inhibits the growth of various cancer cells is currently beinginvestigated.

It will be appreciated that other known agents having antiproliferativeor anti-cancer properties may be combined with the present compositionto enhance its anti-proliferative/anti-cancer activity, including, butnot limited to: alkylating agents such as cisplatin, nitrosoureas suchas carmustine, antimetabolites such as 5-fluoruracil, methotrexate,anthracyclines such as daunorubicin and doxorubicin, topoisomerase IIinhibitors such as topotecan, and mitotoic inhibitors such as taxanes(paclitaxel, docetaxel) and the vinca alkaloids (vinblastine,vincristine, and vinorelbine). In addition, combinations with antibodiesare also envisioned such as trastuzumab, imatinib, gefitinib, erlotinib,rituximab, and bevacizumab.

Example 19

To evaluate the anti-inflammatory effects of the blackberry extract ofthe present invention, bone marrow cells were obtained by flushing thefemurs of BALB/c mice (Harlan Sprague-Dawley Laboratories, Indianapolis,Ind.) with 1×HBSS. Cells were cultured in 100 mm bacteriological petridishes at 2×10⁵ cells/mL in 10 mL of complete RPMI 1640 medium(supplemented with 10% heat-inactivated fetal calf serum, 1 mM HEPES, 2_(C)M L-glutamine, 10 U/mL penicillin, 100 U/mL streptomycin, 50 _(C)M2-mercaptoethanol) containing 20-25 ng/mL GM-CSF at 37° C., 7% CO₂. Thecells were supplemented with an additional 10 mL of complete RPMI 1640with 20-25 ng/mL GM-CSF on day 3. On day 6, 10 mL of supernatant wasremoved from each plate and spun down. The cells were resuspended infresh 10 mL of complete RPMI 1640 with 20-25 ng/mL GM-CSF and added backto the Petri dishes. Non-adherent to lightly adherent cells wereharvested on day 7 as dendritic cells (DCs) and used for the in vitrostudies. For the interleukin-12 (IL-12) release assay, day 7 harvestedbone marrow derived dendritic cells (BMDDCs) were plated in 200 cl ofcomplete RPMI 1640 containing 20-25 ng/mL GM-CSF at 4×10⁵ cells/well in48-well tissue culture plates (Costar) at 37° C., 7% CO₂ overnight. Themedia was removed and replaced with fresh complete RPMI 1640.

Blackberry extract prepared as described in Example 3 was then added inconcentrations providing from 5.1 to 37.3 μg monomeric anthocyanins/mLmedia and plates were incubated for 30 min. High-dose (10 μg/mL) orlow-dose (0.1 μg/mL) Lipid A from Salmonella Minnesota R595 (Re) (ListBiological Laboratories, Campbell, Calif.) was then added to each wellwith or without the berry extract treatment. After 24 hr, supernatant ineach well was collected and stored at −80° C. until IL-12 measurement.Total IL-12 concentration in supernatant was measured using a murinetotal IL-12 ELISA Kit (Pierce) according to the instructions from themanufacturer. Results are presented in FIG. 9.

Previous studies by Pergola and colleagues demonstrated that part of theanti-inflammatory activity of a specific blackberry extract was due tothe suppression of nitric oxide (NO) production in J774 cells bycyanidin-3-O-glucoside (Pergola et al., 2006). Rossi et al. (2003)showed that the anthocyanin fraction from blackberry extract exertedmultiple protective effects in carrageenan-induced pleurisy in rats.Nevertheless, most of the in vitro studies utilizinganthocyanin-containing extracts from other fruits or vegetables havefocused on the effect of the extracts on NO synthesis and TNF-α levelsin vitro using activated macrophages (Wang and Mazza, 2002a; Wang andMazza, 2002b; Hou et al., 2005). Other studies have assessed the effectsof extracts on the inflammation induced by hydrogen peroxide and TNF-ain human microvascular endothelial cells (Youdim et al., 2002). To ourknowledge, there have been few or no studies assessing theanti-inflammatory effects of anthocyanin-containing extracts ondendritic cells. DCs are potent antigen-presenting cells and function asinitiators and modulators of the immune response. Lipid A is known toinduce maturation of DCs resulting in synthesis of high levels ofpro-inflammatory IL-12 that enhances both innate (natural killer cell)and acquired (B and T cells) immunity.

As shown in FIG. 9A, baseline release of IL-12 from non-stimulated DCswas low, with an IL-12 concentration of only 1.46 ng/mL. However, forall concentrations of the berry extract added, the IL-12 release wasreduced in a concentration dependent manner with only 0.37 ng IL-12/mLsecreted using a concentration of 37.3 μg monomeric anthocyanins/mLmedium. As expected, both high-dose (10 μg/mL) and low-dose (0.1 μg/mL)lipid A resulted in very high release of IL-12 from DCs of 624 ng/mL and468 ng/mL, respectively (FIG. 9B). In the low dose lipid A treatmentgroup, the concentration of IL-12 in cell culture supernatant wasdecreased in a concentration dependent manner from 468 to 72 ng/mL whenthe berry extract was added in the range from 5.1 to 37.3 _(C)gmonomeric anthocyanin/mL. A similar pattern was observed in thehigh-dose lipid A treatment group, with the reduction of IL-12 releaseranging from 474 to 138 ng/mL when the berry extract was added in therange from 5.1 to 37.3 μg monomeric anthocyanin/mL. Thus, the berryextract significantly inhibited the release of IL-12 from murine BMDDCswith or without Lipid A treatment. These results suggest that the berryextracts of the present invention may have significant anti-inflammatoryproperties, mediated at least in part by reduced production of certaininflammatory cytokines.

Example 20

To evaluate the ability of various cryoprotectants to stabilizeanthocyanins in berry extracts, a blackberry extract was prepared fromblackberry puree (Example 1) with the extraction method described inExample 3. The cryoprotectants evaluated were:

-   -   1. Dextran: Sigma, 6 kD, Cat#9004-54-0, Lot#1288265;    -   2. Polyvinyl pyrrolidone (PVP): Sigma, 10 kD, Cat#9003-39-8,        Lot#108K0157;    -   3. Carboxymethyl cellulose (CMC): Sigma, 90 kD, Cat#419273, Lot#        MKBF8492V;    -   4. Polyethylene glycol (PEG): Sigma, 8 kD, Cat#89510,        Lot#1394369;    -   5. Lactose: Spectrum, Cas#63-42-3, Lot#V00335;    -   6. Sucrose: Fisher, Cat# BP220-1, Lot#037621;    -   7. Trehalose: Sigma, Cat# TO167, Lot#020M70073V; and    -   8. Hydroxyethyl starch (HES): Sigma, Cat#H6382, Lot#093H0405V,        MW>1 million.        The experimental groups evaluated were: 1) extract powder; 2)        cryoprotectant-extract (2:1, w/w) powder; and 3)        cryoprotectant-extract (8:1, w/w) powder groups.

A 10% aqueous solution of each cryoprotectant was prepared. For thecarboxymethyl cellulose, a 5% aqueous solution was prepared. A stockblackberry extract solution was prepared (140 mg extract powder/mL). Amixture of 10% (v/v) cryoprotectant solution (5% for carboxymethylcellulose) in blackberry extract stock solution (140 mg/mL) was preparedto provide a final cryoprotectant powder:extract powder ratio (w/w) of2:1 and 8:1.

To evaluate anthocyanin stability over time, 10 mL samples (1mL/sample×3 repetitions×3 time points=9 mL) of each solution wereprepared for each of the above cryoprotectants. The final concentrationsof extract in each extract-cryoprotectant solutions were 36.82 mg/mL and11.48 mg/mL for cryoprotectant:berry extract ratios (w/w) of 2:1 and8:1, respectively.

Next, stability of anthocyanins during storage at ambient temperaturewas evaluated according to the following procedure:

-   -   1. One (1) mL aliquots of extract-cryoprotectant or extract        solution (without cryoprotectant) were freeze-dried and the        freeze-dried powder were stored at room temperature (22-28) in        20 ml glass scintillation vials sealed with white polypropylene        screw caps (Fisher, cat# 03-341-25E).    -   2. Total Anthocyanin content (TA) was determined using a pH        differential method [37.1.68    -   AOAC Official Method 2005.02, J. AOAC Int. 88, pg 1269 (2005),        incorporated herein by reference in its entirety] at days 0, 14,        and 28.    -   3. A total of 18 samples were evaluated for each cryoprotectant        [3 time points 2 concentrations 3 repetitions (n=3)=18 samples].        -   Total samples tested at each time point=2 concentrations of            cryoprotectant 8 samples 3 repetitions (n=3)+3 repetition of            pure extract=51.        -   Total samples in this experiment=51×3 time points=153.    -   4. Retention of TA at day 14 and 28 was calculated as percentage        of the TA of each corresponding cryoprotectant or pure extract        group as measured at day 0.

The results are presented in Tables 11-19 below.

TABLE 11 Retention of TA over time at 22-28° C. for berry extract powderwithout cryoprotectant. Extract Alone Day 0 Day 14 Day 28 100.00 ± 6.8688.04 ± 2.20 84.15 ± 4.03

TABLE 12 Retention of TA over time at 22-28° C. for berryextraet-Trehalose powder. Trehalose:Extract (w/w) Day 0 Day 14 Day 282:1 100.00 ± 8.21  103.59 ± 6.56 107.76 ± 3.44 8:1 100.00 ± 10.47 105.12± 2.51  99.26 ± 9.10

TABLE 13 Retention of TA over time at 22-28° C. for berryextract-Lactose powder. Lactose:Extract (w/w) Day 0 Day 14 Day 28 2:1100.00 ± 5.82 101.45 ± 4.34  99.37 ± 2.36 8:1 100.00 ± 6.72 101.82 ±5.94 100.59 ± 3.47

TABLE 14 Retention of TA over time at 22-28° C. for berryextract-Sucrose powder. Sucrose:Extract (w/w) Day 0 Day 14 Day 28 2:1100.00 ± 5.21  96.28 ± 1.12  99.37 ± 2.36 8:1 100.00 ± 5.97 101.93 ±3.63 101.55 ± 1.90

TABLE 15 Retention of TA over time at 22-28° C. for berryextract-Dextran powder. Dextran:Extract (w/w) Day 0 Day 14 Day 28 2:1100.00 ± 6.45  97.97 ± 8.24 101.70 ± 12.41 8:1 100.00 ± 7.51 100.58 ±8.15 100.93 ± 5.61 

TABLE 16 Retention of TA over time at 22-28° C. for berry extract-PVPpowder. PVP:Extract (w/w) Day 0 Day 14 Day 28 2:1 100.00 ± 9.52  91.36 ±0.35  86.73 ± 4.35 8:1 100.00 ± 7.69 101.10 ± 7.13 100.69 ± 7.82

TABLE 17 Retention of TA over time at 22-28° C. for berry extract-HESPowder. HES:Extract (w/w) Day 0 Day 14 Day 28 2:1 100.00 ± 3.36  96.93 ±3.21 91.98 ± 1.70 8:1 100.00 ± 6.72 103.83 ± 4.83 93.82 ± 7.76

TABLE 18 Retention of TA over time at 22-28° C. for berry extract-PEGpowder. PEG8000:Extract (w/w) Day 0 Day 14 Day 28 2:1 100.00 ± 7.61 97.12 ± 11.64 80.36 ± 3.24 8:1 100.00 ± 10.19 75.87 ± 5.36  63.95 ± 3.93

TABLE 19 Retention of TA over time at 22-28° C. for berry extract-CMCpowder. CMC:Extract (w/w) Day 0 Day 14 Day 28 2:1 100.00 ± 5.74 99.12 ±10.09 102.63 ± 5.14  8:1 100.00 ± 6.86 92.15 ± 7.13  92.58 ± 13.69

As clearly demonstrated in Tables 11-19, with the exception of PEGincreased retention of Total Anthocyanin content, i.e., improvedanthocyanin stability, was observed in all berry extract-cryoprotectantpowder compositions tested as compared to berry extract powder withoutcryoprotectant.

It will therefore be appreciated that the present invention provides asimple, convenient method for preparing a berry extract having astabilized anthocyanin content, for use in compositions providing healthand medical benefits to individuals consuming them. The foregoingdescription of the preferred embodiment of this invention has beenpresented for purposes of illustration and description. It is notintended to be exhaustive or to limit the invention to the precise formdisclosed. Obvious modifications or variations are possible in light ofthe above teachings. The embodiment was chosen and described to providethe best illustration of the principles of the invention and itspractical application to thereby enable one of ordinary skill in the artto utilize the invention in various embodiments and with variousmodifications as are suited to the particular use contemplated. All suchmodifications and variations are within the scope of the invention asdetermined by the appended claims when interpreted in accordance withthe breadth to which they are fairly, legally, and equitably entitled.

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1. A stabilized anthocyanin-containing berry powder having antioxidantand anti-inflammatory activity, wherein the berry powder is prepared bythe steps of: i) physically disrupting a quantity ofanthocyanin-containing berries; ii) exposing the physically disruptedberries to an acidic solvent composition having a pH of from about 1 toabout 3 to provide an acidic extract of berries; iii) adding aneffective amount of a cryoprotectant to provide a stabilizedanthocyanin-containing berry extract; and iv) forming the stabilizedanthocyanin-containing berry extract into a berry powder.
 2. Thecomposition of claim 1, wherein the cryoprotectant is selected from thegroup consisting of a monosaccharide, a disaccharide, a polysaccharide,a polymer, and combinations thereof.
 3. The composition of claim 2,wherein the cryoprotectant is selected from the group consisting ofglucose, fructose, maltose, ribose, mannose, and xylose, trehalose,sucrose, myoinositol, phosphorylated inositols, and glycerol,hydroxyethyl starch and other starches, dextran, and hyaluronic acid,polyvinylpyrrolidone, alginates, carrageenan, cyclodextrins, polyvinylalcohol, cellulose-derivatives such as carboxymethyl cellulose, methylcellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose,xanthan gum, chitosan, and combinations thereof.
 4. The composition ofclaim 1, wherein the anthocyanin-containing berry extract is derivedfrom a blackberry.
 5. The composition of claim 1, wherein theanthocyanin-containing berry extract is brought to a pH of from about1.0 to about 4.5.
 6. The composition of claim 1, wherein theanthocyanin-containing berry extract is brought to a pH of about 3.5. 7.The composition of claim 1, wherein the solvent composition comprises analcohol and at least one acid.
 8. The composition of claim 7, whereinthe alcohol is a lower alcohol.
 9. The composition of claim 8, whereinthe lower alcohol is selected from the group consisting of methanol,ethanol, propanol, butanol, and combinations thereof.
 10. Thecomposition of claim 7, wherein the at least one acid is selected fromthe group consisting of hydrochloric acid, acetic acid, citric acid,lactic acid, trifluoroacetic acid, aspartic acid, glutamic acid, asulfur-containing acid, sulfonic acid, formic acid, phosphoric acid,maleic acid, and combinations thereof.
 11. The composition of claim 1,wherein the physically disrupted anthocyanin-containing berries aredewatered to a water content of up to 20% (w/v) prior to or afterexposing to the solvent composition.
 12. The composition of claim 11,wherein the cryoprotectant is added in an amount of at least 2:1 (w/w)cryoprotectant:anthocyanin-containing berry extract.
 13. The compositionof claim 1, wherein the anthocyanin-containing berry extract is providedas a formulation adapted for oral administration.
 14. The composition ofclaim 13, wherein the anthocyanin-containing berry extract is providedas a nutritional supplement, a capsule, an enteric-coated capsule, afilm-coated capsule, a tablet, an enteric-coated tablet, a film-coatedtablet, or a chewing gum.
 15. The composition of claim 14, comprisingthe anthocyanin-containing berry extract in an amount of from about 3%(w/w) to about 90% (w/w).
 16. The composition of claim 1, wherein theanthocyanin-containing berry extract is provided as a formulationadapted for topical administration.
 17. The composition of claim 16,wherein the anthocyanin-containing berry extract is provided as alotion, a cream, a mucoadhesive gel, a vanishing lotion, or a vanishingcream.
 18. The composition of claim 17, comprising theanthocyanin-containing berry extract in an amount of from about 1% (w/w)to about 20% (w/w).
 19. A composition for treating inflammation,oxidative damage, or cancer in a mammal in need thereof, comprising atherapeutically effective amount of a composition as recited in claim 1in a pharmaceutically acceptable vehicle.
 20. A method for preparing astabilized anthocyanin-containing berry powder having antioxidant andanti-inflammatory activity, comprising: physically disrupting a quantityof anthocyanin-containing berries; exposing the disrupted berries to anacidic solvent composition having a pH of from about 1 to about 3;adding an effective amount of a cryoprotectant; and forming thestabilized anthocyanin-containing berry extract into a berry powderhaving a stabilized anthocyanin content.
 21. The composition of claim20, including selecting the cryoprotectant from the group consisting ofa monosaccharide, a disaccharide, a polysaccharide, a polymer, andcombinations thereof.
 22. The method of claim 21, including selectingthe cryoprotectant from the group consisting of glucose, fructose,maltose, ribose, mannose, and xylose, trehalose, sucrose, myoinositol,phosphorylated inositols, and glycerol, hydroxyethyl starch and otherstarches, dextran, and hyaluronic acid, polyvinylpyrrolidone, alginates,carrageenan, cyclodextrins, polyvinyl alcohol, cellulose-derivativessuch as carboxymethyl cellulose, methyl cellulose, hydroxypropylcellulose, hydroxypropylmethyl cellulose, xanthan gum, chitosan, andcombinations thereof.
 23. The method of claim 20, wherein theanthocyanin-containing berry is a blackberry.
 24. The method of claim20, including the step of exposing the physically disruptedanthocyanin-containing berries to the solvent composition and recoveringan anthocyanin-containing berry extract having a pH of from about 1.0 toabout 4.5.
 25. The method of claim 24, including recovering ananthocyanin-containing berry extract having a pH of about 3.5.
 26. Themethod of claim 24, including exposing the physically disruptedanthocyanin-containing berries to a solvent composition comprising analcohol and at least one acid.
 27. The method of claim 26, includingexposing the physically disrupted anthocyanin-containing berries to asolvent composition comprising a lower alcohol and at least one acid.28. The method of claim 27, including selecting the lower alcohol fromthe group consisting of methanol, ethanol, propanol, butanol, andcombinations thereof.
 29. The method of claim 27, including selectingthe at least one acid from the group consisting of hydrochloric acid,acetic acid, citric acid, lactic acid, trifluoroacetic acid, asparticacid, glutamic acid, a sulfur-containing acid, sulfonic acid, formicacid, phosphoric acid, maleic acid, and combinations thereof.
 30. Themethod of claim 24, further including the step of dewatering thephysically disrupted anthocyanin-containing berries to a water contentof up to 20% (w/v) prior to or after the step of exposing to the solventcomposition.